Room temperature stable agarose solutions

Abstract

A method for making functionalized porous crosslinked polysaccharide gel coated structures used as liquid chromatography media is provided. The method includes impregnating a porous substrate with a room temperature stable aqueous polysaccharide solution containing water, 0.1% to 20% of a polysaccharide, 18% to 54% of a gel-inhibiting agent to prevent the gel from re-gelling, and 0.001% to 10% of an anionic fluorosurfactant for optimum solution coatability onto the substrate, each concentration is by total weight of the aqueous solution. Next water is evaporated from the coating, followed by exposing the dehydrated coating to a gelling agent thereby forming a porous polysaccharide gel coated substrate. Next the gel coated substrate is exposed to a crosslinking agent forming a porous crosslinked polysaccharide gel coated substrate. Next, the gel coated substrate is functionalized by contacting the porous polysaccharide gel coated substrate with sodium 3-bromopropanesulfonate, thereby attaching sulfopropyl ligands to the gel coated substrate, resulting in a functionalized porous absorptive polysaccharide gel coated chromatography structure. When the functionalized gel coated chromatography structure is used as liquid chromatography media, and a protein (e.g. lysozyme) containing solution contacts the media, the sulfopropyl ligands attached to the gel coated chromatography structure will bond to the protein, thereby removing the protein from the protein containg solution.

The invention claimed is: 
     
       1. A method for making a porous functionalized crosslinked polysaccharide hydrogel coated structure comprising:
 a) impregnating a porous substrate selected from the group consisting of fibers, woven fabrics, nonwoven fabrics, membranes, mats, monolithic honeycomb structures, felt sheets, poly(HIPES) monolithic structures, three dimensional structures, sponges, and porous beads, with a room temperature stable aqueous polysaccharide solution forming a room temperature stable aqueous polysaccharide coating on the substrate, the aqueous solution containing, i) water, ii) a polysaccharide having a concentration in the aqueous solution from about 0.1% to about 20%, and selected from the group consisting of agarose, dextrans, cyclodextrins and blends thereof, iii) a gel-inhibiting agent having a concentration in the aqueous solution from about 18 to about 54%, and selected from the group consisting of ZnCl 2 , LiCl, NaOH, LiNO 3 , LiOH, LiSCN and mixtures thereof, and iv) an anionic fluorosurfactant having a concentration in the aqueous solution from about 0.001% to about 10% by total weight of the aqueous solution; 
 b) evaporating the water from the polysaccharide coating; 
 c) exposing the dehydrated polysaccharide coating to a gelling agent selected from the group consisting of acetone and acetone in water to form a porous polysaccharide hydrogel coated substrate: 
 d) exposing the hydrogel coated substrate to a crosslinking agent selected from the group consisting of epichlorohydrin, multifunctional epoxy compounds, multifunctional halide compounds, formaldehyde, gluteraldehyde, multifunctional aldehyde compounds, bis(2-hydroxy ethyl)sulfone, dimethyldichlorosilane, dimethylolurea, dimethylol ethylene urea, diisocyanate compounds, and polyisocyanate compounds to form a porous crosslinked polysaccharide hydrogel coated substrate; and 
 e) contacting the crosslinked polysaccharide hydrogel coated substrate with sodium 3-bromopropanesulfonate, and attaching sulfopropyl functional groups by a functionalization reaction to the crosslinked polysaccharide hydrogel coated substrate. 
 
     
     
       2. The method according to  claim 1 , wherein the porous substrate is a polyolefin non-woven fabric. 
     
     
       3. The method according to  claim 1 , wherein the polysaccharide is agarose. 
     
     
       4. The method according to  claim 1 , further comprising a step of rinsing the crosslinked polysaccharide hydrogel coated substrate with water between steps (d) and (e). 
     
     
       5. The method for making a functionalized liquid chromatography media comprising:
 a) impregnating a porous substrate selected from the group consisting of fibers, woven fabrics, non-woven fabrics, membranes, mats, monolithic honeycomb structures, felt sheets, poly(HIPES) monolithic structures, three dimensional structures, sponges, and porous beads, with a room temperature stable aqueous agarose solution, the aqueous agarose solution containing, i) water, ii) agarose having a concentration in the aqueous solution from about 0.1% to about 20%, iii) a gel-inhibiting agent having a concentration in the aqueous solution from about 18 to about 54%, and selected from the group consisting of ZnCl 2 , NaOH, LiNO 3 , LiOH, LiSCN and mixtures thereof, and iv) an anionic fluorosurfactant having a concentration in the aqueous solution from about 0.001% to about 10% by total weight of the aqueous solution; 
 b) forming a room temperature stable aqueous agarose coating on the substrate; 
 c) evaporating the water from the agarose coating; 
 d) exposing the agarose coating to a gelling agent selected from the group consisting, of acetone and acetone in water to form a porous polysaccharide hydrogel coated substrate; 
 e) exposing the hydrogel coated substrate to a crosslinking agent selected form the group consisting of epichlorohydrin, multifunctional epoxy compounds, multifunctional halide compounds, formaldehyde, gluteraldehyde, multifunctional aldehyde compounds, bis(2-hydroxy ethyl)sulfone, dimethyldichlorosilane, dimethylolurea, dimethylol ethylene urea, diisocyanate compounds, and polyisocyanate compounds to form a porous crosslinked agarose hydrogel coated substrate; 
 f) rinsing the porous crosslinked agarose hydrogel coated substrate with water, and 
 g) contacting the crosslinked agarose hydrogel coated substrate with odium 3-bromopropanesulfonate, and attaching sulfopropyl functional groups by a functionalization reaction to the porous crosslinked agarose hydrogel hydrogel coated substrate forming, a functionalized liquid chromatography media. 
 
     
     
       6. The method according to  claim 5 , wherein the porous substrate is a polyolefin non-woven fabric. 
     
     
       7. A functionalized liquid chromatography media comprising:
 a) a porous substrate selected from the group consisting of fibers, woven fabrics, non-woven fabrics, membranes, mats, monolithic honeycomb structures, felt sheets, poly(HIPES) monolithic structures, three dimensional structures, sponges, and porous beads; 
 b) a porous crosslinked agarose hydrogel coating located on the porous substrate, the crosslinked agarose hydrogel coating formed from, a room temperature stable aqueous agarose solution applied to the substrate containing, i) water, ii) agarose having a concentration in the aqueous solution from about 0.1% to about 20%, iii) a gel-inhibiting agent having a concentration in the aqueous solution from about 18 to about 54%, and selected from the group consisting of ZnCl 2 , LiCl, NaOH, LiNO 3 , LiOH, LiSCN and mixtures thereof, and iv) an anionic fluorosurfactant having a concentration in the aqueous solution from about 0.001% to about 10% by total weight of the aqueous solution, wherein the water is evaporated from the coating and the coating is exposed to v) a gelling agent selected from the group consisting of acetone and acetone in water forming a porous agarose hydrogel coated substrate, wherein the porous agarose hydrogel coated substrate is exposed to vi) a crosslinking agent selected form the group consisting of epichlorohydrin, multifunctional epoxy compounds, multifunctional halide compounds, formaldehyde, gluteraldhyde, multifunctional aldehyde compounds, bis(2-hydroxy ethyl)sulfone, dimethyldichlorosilane, dimethylolurea, dimethylol ethylene urea, diisocyanate compounds, and polyisocyanate compounds forming a porous crosslinked agarose hydrogel coated substrate: and 
 c) contacting the crosslinked agarose hydrogel coated substrate with sodium 3-bromopropanesulfonate, and attaching sulfopropyl functional groups by a functionalization reaction to the porous crosslinked agarose hydrogel coated substrate. 
 
     
     
       8. The chromatography media according to  claim 7 , wherein the porous substrate is a polyolefin non-woven fabric.